Inhibition of microRNA-33b in humanized mice ameliorates nonalcoholic steatohepatitis

Experiments using humanized mice created by knocking in microRNA (miR)-33b into an intron of Srebf1 show that suppression of miR-33b by nucleic acid drugs is useful for the treatment of NASH.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** ---------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): In this study, Sawa identified the role of miR-33b in NASH. Due to the absence of miR-33b in mice, the authors generated miR-33b knock-in mice. By using this KI mice, they found that hepatocyte-specific not macrophage-specific miR-33b deletion ameliorated NASH-reltaed fibrosis and inflammation. Importantly, anti-miR-33b treatment limited NASH, suggesting that targeting miR-33b in hepatocytes provides a novel strategy for treatment of NASH. Although this work is interesting, more evidence are needed to support the conclusion. 1.miR-33b is abundantly expressed in human not in rodents. It would be interesting to add evidences from NASH patients for clinical correlation and significance of this study. 2.New targets of miR-33b were not found in this study. The authors shoud try to find a new target. 3.More experiments are needed to suppot that anti-miR-33b ameliorates NASH-related inflammation. For example, F4/80 and MPO staining.
Reviewer #2 (Comments to the Authors (Required)): In their manuscript Miyagawa et al show that genetically engineered mice expressing miR33b within their Srebf1 gene, as naturally occuring in humans, develop steatosis (NAFLD) and features of steatohepatitis (NASH) when fed a high-fat diet (HFD) or Gruba-Amylin NASH diet (GAN). They further attribute this effect to hepatocyte-specific miR33b expression, and not macrophage, through the use of a cell-specific Cre-inducible miR33b knock-in system. Finally, they show that silencing of miR33b but not miR33a via anti-miR oligonucleotide (AMO) administration in miR33b KI mice was sufficient to prevent NAFLD/NASH development in mice showing the specificity of miR33b activity in this phenotype.
The authors have performed a well-rounded study, using what could be described as a top-down approach where they leveraged the power of murine models for in vivo testing of what would be an otherwise primate-specific gene. They successfully generated a Cre-inducible KI of miR33b, used two different models of diet-inducible NAFLD and NASH, and analyzed the development or regression of the phenotype via gold-standard metrics. The use of AmNA-prepared AMO successfully showed specificity between miR33a and miR33b, despite a difference of 2 nucleotides between the two sequences is another impressive feature of the manuscript. Overall, these studies are well performed, the data are clearly presented and the manuscript is wellwritten.
1. miR33a and miR33b have both been linked to NASH in humans already (Erhartova D. 2019; Auguet T. 2016) and they have the same mRNA targets, as published by the same group (Horie T. 2014). Their effect on NASH seems to rely on the preferential expression of Srebf1 over Srebf2 in this murine model, thereby leading to a higher expression of miR33b. However, the transcriptional activity of SREBPs in humans is known to be distinct from mice and this should be discussed as a limitation of the study.
For instance, in humans, there are reports of SREBP2 but not SREBP1c upregulation in patients with NASH compared to simple steatosis (Caballero F. J Hepatol. 2009), with other reports showing protein but not mRNA upregulation of SREBF2 in NASH, but not NAFLD or obesity (Min HK. Cell Metab. 2012). On the other hand, a moderate increase in SREBF1 has been found in patients with steatosis (below 2-fold), but not NASH (Dorn C. Int J Clin Exp Pathol. 2010). This has been replicated in a cohort of patients with obesity and steatosis but not NASH, with an increase below 2-fold again (Petinelli P. JCEM. 2011). More recently, upstream transcriptional activation analysis by next-generation sequencing in biopsy-proven NASH suggests that both SREBF1 and SREBF2 are active, but with no demonstration of increased transcription of the SREBF1 or SREBF2 locus (Azzu V. Mol Metab. 2021). These findings bring the relevance of the current study into question.
Whether different nutrients in the diets could influence the phenotype should also be discussed. The work of Jay Horton on the contribution of the Srebf2 pathway, and thus of cholesterol flux, to the generation of LXR agonists required for SREBP1c transcriptional activity suggest that differences in cholesterol content in the diet influences the cooperation of both transcription factor, which could also explain differences between human pathology and murine models of diet-induced NAFLD/NASH.

Response to Reviewer #1
We are grateful to Reviewer #1 for the informative and useful comments. As described below, we have considered all of these comments and used them to improve our manuscript.

Reviewer #1:
In this study, Sawa identified the role of miR-33b in NASH. Due to the absence of miR-33b in mice, the authors generated miR-33b knock-in mice. By using this KI mice, they found that hepatocyte-specific not macrophage-specific miR-33b deletion ameliorated NASH-reltaed fibrosis and inflammation. Importantly, anti-miR-33b treatment limited NASH, suggesting that targeting miR-33b in hepatocytes provides a novel strategy for treatment of NASH.
Although this work is interesting, more evidence are needed to support the conclusion.

1.miR-33b is abundantly expressed in human not in rodents. It would be interesting to add evidences from NASH patients for clinical correlation and significance of this study.
Thank you very much for your valuable comments. We cited previous papers on human NASH and explained the importance of the present results in NASH patients. We also discussed the possibility that SREBF1 and SREBF2, which are thought to be expressed similarly to miR-33b and miR-33a, may behave differently in mice and humans, and added this point to Discussion.

Inserted sentence (on page 22, paragraph 4, lines 27-page 23, lines 13):
There are several limitations of the study in this paper.  2017), differences in diet may create differences between human NASH and mouse models.

2.New targets of miR-33b were not found in this study. The authors shoud try to find a new target.
Thank you for very much for your comments. The most important points of this paper are that mice with the same miR-33b as humans show NASH on a high-fat, high-cholesterol diet and that nucleic acid drugs targeting miR-33a/b are effective against NASH for which there are currently no effective treatments. Although no new miR-33a/b target genes were found in the current paper, we believe that the robustness of the previous papers has been proven. Inserted sentence (on page 18, paragraph 1, lines 5-8): In addition, F4/80 and MPO staining was also performed to determine the effect of anti-miR-33a and anti-miR-33b on inflammation. The results confirmed that the GAN diet increased F4/80 and MPO-positive cells in the liver, while administration of anti-miR-33a and anti-miR-33b decreased them (Supplementary figure 7).

Response to Reviewer #2
We are grateful to Reviewer #2 for the informative and useful comments. As described below, we have considered all of these comments and used them to improve our manuscript. Whether different nutrients in the diets could influence the phenotype should also be discussed.

The work of Jay Horton on the contribution of the Srebf2 pathway, and thus of cholesterol flux, to the generation of LXR agonists required for SREBP1c transcriptional activity suggest that differences in cholesterol content in the diet influences the cooperation of both transcription
factor, which could also explain differences between human pathology and murine models of

diet-induced NAFLD/NASH.
Thank you very much for your comment. As you indicated, based on previous papers, there may be differences in the behavior of SREBF1 and SREBF2 between mice and humans, and the following sentence has been added to Discussion as a limitation of this paper.  2017), differences in diet may create differences between human NASH and mouse models.

Given the concerns raised in point #1
, the title should be revised to more accurately represent the work presented by including "humanized mice" -Inhibition of miR-33b in humanized mice ameliorates nonalcoholic steatohepatitis Thank you for your comment. We have changed the title of our manuscript as you suggested.
3. The discussion should also be reorganized to discuss the difference in copy numbers between miR33a and miR33b. Deficiency in miR33a has been implicated in NAFLD/NASH development in mice by the same group (Horie T. 2013), why is targeting of miR33a with AMO not reproducing this phenotype?
Thank you very much for your comments. Recently we have identified the cause of obesity in miR-33a-deficient mice (Nat Commun. 2021 Feb 16;12(1):843.). Our results suggest that loss of miR-33a action in the hypothalamus in miR-33a-deficient mice results in inactivation of sympathetic nerves and brown adipose tissue, leading to obesity in these mice. Since the anti-miR-33a in this paper does not cross the blood-brain barrier, obesity due to miR-33a suppression does not occur.  Thank you for submitting your revised manuscript entitled "Inhibition of microRNA-33b in humanized mice ameliorates nonalcoholic steatohepatitis". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.

In the introduction, the authors write that miR-33a inhibition
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